1. Field of the Invention
This invention relates generally to methods for controlled deposition of polymers (e.g., polysaccharides), and optionally for coupling molecules, including biomolecules, cellular species, and the like to the polysaccharides in deposition. This invention further relates to materials, such as hydrogels, and to devices comprising electrochemically deposited polysaccharides, alone or in combination with coupled molecules.
2. Description of Related Art
The use of micro-electro-mechanical systems (MEMS) in biological research is becoming increasingly common. Micro-devices allow for relatively easy observation and manipulation of individual cells, proteins, or other biological macromolecules. Sample sizes for such experiments may be reduced when using MEMS as compared to traditional techniques. J. D. Trumbull, et al., IEEE Transactions on Biomed. Eng. 47, 3 (2000). This allows biological systems to be studied at a new level of resolution while minimizing the materials required for an experiment.
Initially, microfluidic devices were used primarily for capillary electrophoresis. S. Jacobson, et al., Anal. Chem 66 (1994) 1114; D. J. Harrison, et al., Anal. Chem. 64 (1992) 1926; Z. Liang, et al.; Anal. Chem. 68 (1996) 1040. Recently, there has been interest in incorporating a complete array of functional units, e.g., valves, pumps, reaction chambers, etc., onto a single chip to create a lab-on-a-chip (LOC). J. Voldman, et al., J. Microelectromech. Sys. 9 (2000) 295; I. Glasgow, et al., IEEE Transactions on Biomed. Eng. 48 (2001) 570; T. Fujii, Microelectronic Eng., 61-62 (2002) 907; A. Yamaguchi, et al., Analytica Chimica Acta., 468 (2002) 143; J. H. Kim, et al., Sensors and Actuators A. 95 (2002) 108; M. Krishnan, et al., Curr. Opinion Biotech. 12 (2001) 92; A. Hatch, et al., J. Microelectromech. Sys. 10 (2002) 215.
The ability to create MEMS and other devices such as biosensors and microarrays requires facile methods to precisely control surfaces. A variety of patterning techniques can be used to produce desired structures, while various methods have been investigated to control surface chemistries. For instance, microfabrication techniques are routinely applied to create patterned inorganic surfaces with nanometer to micrometer scale resolution. Xia, Y, et al., Angew. Chem, Int. Ed. Engl., 37, 550-575 (1998).
Two approaches have emerged to extend microfabrication techniques for the creation of patterned surfaces with organic and biological materials. The first approach is based on an extension of photolithography. Bain, C. D., et al., Angew. Chem., Int. Ed. Engl., 28, 506-512 (1989); Whitesides, G. M., Langmuir, 6, 87-96 (1990). Self-assembled monolayers are selectively irradiated to create a pattern of freshly exposed surface, which is then reacted with a bifunctional agent. Reactions include those between thiols and metal surfaces, or between silanes and oxidized silicon. Bain, C. D., et al., Chem. Int. Ed. Eng/. 1989,28, 506-512; Whitesides, G. M.,et al., Langm., 6, 87-96 (1990); Sagiv, J. J. Am. Chem. Soc. 102, 92-98 (1980); Brzoska, J. B., et al., Langm., 10, 4367-4373 (1994); Allara, D. L., et al., Langm., 11, 2357-2360 (1995).
A first functional group of the agent attaches the agent to the freshly exposed surface, and the second functional group subsequently couples the molecules of interest. Although variations exist, lithography creates the spatial template upon which subsequent coupling occurs. Vossmeyer, T., et al., Angew. Chem., Int. Ed. Engl., 36, 1080-1083 (1997); Vossmeyer, T., et al., J. Appl. Phys., 84, 3664-3670 (1998); Jones, V. W., et al., Anal. Chem., 70, 1233-1241 (1998); Harnett, C. K., Langmuir, 17, 178-182 (2001); Jonas, U., et al., Proc. Natl. Acad. Sci. USA., 99, 5034-5039 (2002). This first approach has a drawback associated with the need for photo-sensitive reagents that can be expensive, hazardous and require cumbersome steps to prepare the surface. Furthermore, conventional photolithographic operations require “line-of-sight” and would be difficult to accomplish on internal surfaces in an enclosed microfluidic system. Alternatively, if the lithographic patterning and subsequent biological functionalization are carried out before the microfluidic device is covered to form a closed fluidic environment, the biofunctionality internal to the microfluidic system cannot be readily reprogrammed. Finally, since many biospecies are labile, i.e., sensitive and delicate with respect to their environmental conditions, fabrication processes required to close the microfluidic system may degrade the biospecies.
A second approach for creating patterned surfaces with organic and biological materials is microcontact printing (μCP), in which a soft stamp (typically made of poly-dimethylsiloxane) is created with a preselected pattern. After “inking” the stamp with a solution containing the material to be deposited, the stamp is pressed onto a surface to transfer the pattern. Xia, Y., et al., Langmuir, 12, 4033-4038 (1996); Hidber, P. C. et al., Langmuir, 12, 1375-1380 (1996). Drawbacks to the microcontact printing approach involve difficulties in stamping with high spatial resolution. Furthermore, the need for direct contact to the surface entails the drawbacks described above for applications to enclosed microfluidic systems. Vaeth, K. M., et al., Langmuir 2000, 16, 8495-8500.
Another approach to patterning biomolecules on surfaces is “dip-pen” nanolithography, in which scanning probe microscopy (like atomic force microscopy) is used to write species onto a surface with high lateral resolution. For biomolecular species this is accomplished by transport from the writing tip through a water meniscus to the substrate. While the lateral spatial resolution of this patterning method can be very high (30 nm), patterns must be written in serial fashion, entailing the throughput limitations associated with other direct-write approaches such as electron and ion beam lithographies. In addition, dip-pen nanolithography entails the drawbacks described above for applications to enclosed microfluidic systems. Piner, R. D., et al., Science 283, 661-663 (1999); Jong, S., Mirkin, C. A., Science, 288, 1808-1811 (2000); Lyuksyutov, S. F. et. al., Nature Materials, 2, 468-474 (July 2003).
Electrophoretic deposition has also been used to assemble colloidal particles and proteins onto electrode surfaces. This approach has been extended to exploit an electric field to direct the spatially selective deposition of CdTe nanocrystals. Gao, M, et al, Langmuir, 18, 4098-4102 (2002). In this method, a surface with patterned electrodes is first fabricated, then a combination of an applied voltage and layer-by-layer assembly is used to generate multilayers with spatial resolution in lateral directions. The drawbacks to this assembly approach are that voltages must be maintained to retain the initial layer of nanocrystals, which may not be held to the surface by strong chemical bonds or insolubility. Again, it is not clear whether these layer-by-layer approaches can be extended to enclosed microfluidic channels.
Another drawback to several of the above approaches is that the deposited film provides a non-aqueous microenvironment that is less appropriate than aqueous environments for some sensitive biological systems. Ito, Y., et al., Langmuir, 13, 2756-2759 (1997). For example, in the case of proteins, a non-aqueous microenvironment may be denaturing, as proteins tend to unfold, when immobilized, which often causes loss of activity and binding sites that may be dependent upon the three-dimension structure. Thus, hydrogels are often considered for applications involving proteins and nucleic acids, and even intact cells. Burns, M. S., et al., Science, 282, 484-487 (1998); Sirkar, et al., Anal. Chem., 70, 2888-2894 (1998); Revzin, A. F., et al., Langmuir, 17, 5440-5447 (2001); Revzin, A. F., et al., Sens. Actuators, 81, 539-568 (2002); Stenger, D. A., et al., Trends Biotechnol., 19, 304-309 (2001); O'Connor, S. M., et al., Biosens. Bioelectron, 14, 871-881 (2000); Reininger-Mack, A., et al., Trends Biotechnol., 20, 56-61 (2002); Koh, W. G., et al., Langmuir, 18, 2459-2462 (2002).